EP3259631B1 - Vorrichtung und verfahren zur erstellung eines optischen tomogramms einer mikroskopischen probe - Google Patents

Vorrichtung und verfahren zur erstellung eines optischen tomogramms einer mikroskopischen probe Download PDF

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EP3259631B1
EP3259631B1 EP15707313.1A EP15707313A EP3259631B1 EP 3259631 B1 EP3259631 B1 EP 3259631B1 EP 15707313 A EP15707313 A EP 15707313A EP 3259631 B1 EP3259631 B1 EP 3259631B1
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sample
lens
microscope
optical
images
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EP3259631A1 (de
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Jan Huisken
Andrea Bassi
Benjamin Schmid
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/12—Condensers affording bright-field illumination
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/26—Stages; Adjusting means therefor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes

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  • the present invention relates to a method for creating an optical tomogram or for creating a tomogram on the basis of optically created section images.
  • the invention further relates to an optical microscope for creating tomograms, which comprises at least one lens, at least one sample holder and at least one light source.
  • optical tomography Another comparatively new method for representing microscopic objects is optical tomography. It makes three-dimensional imaging of objects possible without having to specially pre-treat them, for example using fluorophores.
  • this method requires the sample to be translucent at least in part to light of the wavelength used.
  • For optical tomography it is necessary to record a number of projections of the sample, each from a different angle.
  • this requires the depth of focus of the optics to span the entire depth of the sample. This large depth of focus is generally achieved with optics with low numerical aperture (NA), which limits the achievable resolution of the images and the subsequent reconstruction of the sample.
  • NA numerical aperture
  • the object of the present invention is therefore to provide a method and a device with each of which it is possible to record optical tomograms in a gentle and greatly accelerated manner. This object is achieved by a method according to claim 1 and a device according to claim 12.
  • An essential aspect of the invention is a method for creating an optical tomogram, characterized in that it comprises the following steps:
  • This method makes it possible to accelerate significantly the recording of the section images required for the optical tomography and reduce the burden for living specimens by moving the sample along a smooth trajectory without any abrupt changes in direction.
  • n is usually > 3, preferably > 5, particularly preferably between 10 and 1000.
  • n n
  • the imaging system offers a certain depth of focus.
  • the stack of images is ideally acquired such that the images provide a gap-less coverage of the entire depth.
  • shallow depth of focus (as usually present in high-resolution imaging systems, such as light sheet microscopes) requires more images to be acquired than in cases where the depth of focus is large.
  • n n between 15 and 100 has been found to be particularly preferred.
  • These values of n give a good ratio between resolution and storage requirement or measurement time.
  • the sample after each rotation through the angle ⁇ the sample is displaced by a distance along the longitudinal axis of the lens and/or perpendicular to the plane of the previously recorded image. It is preferably displaced in this direction by a distance of 360 ⁇ D ⁇ ⁇ n , D being the total displacement of the sample during the creation of the optical tomogram. Accordingly, in this method the sample is displaced for each angle by a distance which, after the complete rotation of the sample through 360°, adds to a total distance corresponding to the respective thickness of a layer.
  • the sample is thus imaged in layers in a spiralling manner in each case.
  • the spiral thus formed is preferably an Archimedean spiral, in such a way that the radius varies in proportion to the angle of rotation. Accordingly, at each angle the focal plane is displaced by the constant amount D n .
  • the displacement along the longitudinal axis of the lens is also preferably the length D n , D being the total displacement of the sample during the creation of an optical tomogram.
  • a method variant has been found to be particularly preferred in which the sample is displaced continuously along the longitudinal axis of the lens and/or perpendicular to the plane of the previously recorded image during the recording of at least 3 images, preferably at least 50 images, particularly preferably at least 360 images, from different angles ⁇ .
  • the sample is preferably displaced along the longitudinal axis of the lens continuously throughout the data collection period. The rotation of the sample is superposed on this linear displacement thereof.
  • the sample accordingly moves in a smooth spiral through the focal plane of the lens, which plane - as disclosed above - is preferably not changed throughout the measurement.
  • a method variant has been found to be preferred in which the optical axis of the objective lens is arranged to lie in the horizontal plane.
  • the sample is rotated perpendicular to the longitudinal direction of the lens and more preferably about a vertical axis. This is advantageous because the direction of the gravity acting on individual parts of the sample during the rotation does not vary eliminating any deformations due to gravity.
  • the sample is held substantially vertical.
  • it may either be deposited on a vertically upright support or be hung down into the coverage region of the lens from above.
  • the sample in the optical coverage region of the lens of the microscope, it has been found to be particularly advantageous to hang the sample down into the coverage region of the lens.
  • the sample can be handled and in particular rotated in a very simple manner and largely without friction.
  • the sample is precisely positioned on the rotational axis and in the optical coverage region of the lens of the microscope, preferably by means of micrometer screws arranged on the sample holding system.
  • the sample may also be precisely positioned using other suitable devices.
  • the rotation of the sample and the displacement in the direction of the lens are preferably carried out by actuating and moving the entire sample suspension system accordingly.
  • This usually includes a manipulator, by means of which the sample can be precisely orientated in the coverage region of the lens.
  • the movement is preferably controlled by means of linear or servo motors.
  • the microscope preferably comprises a device for detecting digital images, for example a digital camera or CCD camera.
  • the individual images are therefore captured digitally and saved in a memory. Subsequently, they are preferably combined by a data processing device to form a three-dimensional model. It is advantageous if the calculation of the three-dimensional model of the sample only begins after the first complete rotation of the sample through 360°.
  • the buffer for storing individual images therefore has to be at least large enough to be able to store the number of individual recordings which are to be made during a complete rotation of the sample through 360°.
  • the calculation of the three-dimensional structure preferably takes place in batches as a function of the respective recording angle.
  • a method variant is more strongly preferred in which a bright-field or transmitted-light microscope is used as the optical microscope.
  • a further essential aspect of the invention is an optical microscope for creating tomograms, which comprises at least one lens, at least one sample holder and at least one light source, the sample holder
  • the microscope comprises a data processing device having a memory or in which it is connected to one, in such a way that by means of said device individual images captured digitally by the microscope can be stored and subsequently combined to form a three-dimensional model of the sample.
  • a data processing device of this type may for example be a computer on which corresponding software can be run.
  • Software of this type has to include an algorithm by means of which it is possible to assemble a stack of individual images to form a three-dimensional model.
  • the memory of the optical microscope has a minimum storage capacity which makes it possible to store at least a number i of images which can be captured during a rotation of the sample through 360°, i being the number of individual images recorded during a complete rotation of the sample. If after rotation through an angle ⁇ a section image is captured, i is 360 ⁇ .
  • a further preferred embodiment of the optical microscope is a bright-field microscope in which at least one light source is arranged on the side of the sample opposite the lens.
  • the light source is arranged along the longitudinal axis of the lens but on the opposite side of the sample with respect to the lens.
  • This arrangement of the light source has been found to be particularly advantageous because the provided displacement of the sample along an extension of the longitudinal axis of the lens means that the angle of the incident light in a dark-field or incident-light microscope is forced to change slightly, in such a way that additional artefacts due to altered incident light can occur during recording.
  • a light source may always be arranged on an extension of the longitudinal axis of the lens on the side opposite the sample, and the sample may thus illuminate the sample from the rear face thereof irrespective of the positioning of said sample between the lens and the light source.
  • Fig. 1a is a plan view of a sample 1 prior to positioning on the focal plane 2.
  • the sample 1 is a zebrafish embryo, of which merely a section in the head region is shown in this example.
  • a point or axis is marked about which the sample 1 can be rotated (in direction R).
  • displacement of the sample 1 in the direction of the arrow P is provided.
  • the focal plane 2 is marked by parallel lines 2a, 2b which are intended to indicate the position of the focal plane.
  • the lens 5 is not shown in Fig. 1a . However, it is located on the side of the focal plane 2 opposite the sample 1.
  • An illumination device 6 (also not shown in Fig. 1a ) is arranged on the side of the sample 1 opposite the focal plane 2 with respect to the lens 5.
  • Fig. 1a shows the arrangement of the sample 1 with respect to the focal plane 2 prior to the start of data recording.
  • the sample 1 has not yet been displaced in the direction P of the focal plane, and so there is not yet a layer 4 (see fig. 1c ) of the sample 1 located on the focal plane 2.
  • Fig. 1b is a plan view of a sample 1 during the recording of section images along the focal plane 2.
  • the sample 1 is rotated and displaced in the direction of the arrows P, R.
  • the points 7a - 7z shown in Fig. 1b each represent a particular recording position.
  • Each point 7a - 7z represents a recorded section image which is perpendicular to an imaginary line 8 connecting the respective point 7a - 7z to the axis of rotation 3.
  • the points 7a - 7z are arranged in a spiral, and the arrangement of the focal plane 2 or imaging plane can be seen by way of the example of the point 7z arranged innermost on the spiral.
  • FIG. 1c This shows a sample 1 having a central section plane in the focal plane 2.
  • each of the points 7a - 7z represents a section image which has been recorded from the corresponding angle ⁇ .
  • section images are recorded from each angle ⁇ independently of the displacement of the sample 1.
  • the rectangle 17 drawn in Fig. 1c indicates a stack or batch of section images corresponding to a specific angle ⁇ .
  • Fig. 2 is a schematic drawing of section images 9, arranged in a spiral, of a sample 1.
  • the principle disclosed previously in connection with Fig. 1a - c is illustrated schematically again in Fig. 2.
  • Fig. 2 shows a lens 5 of the microscope which is suitable for sharply imaging a section image 9 of a sample 1 in each case.
  • the focal plane 2 is indicated in Fig. 2 by the axes x, y, which are perpendicular to the axis z representing the longitudinal direction of the lens 5.
  • Fig. 2 shows section image 9 which represent an example of a number of already recorded section images 9. These are arranged along a spiral. This spiral results from the displacement and simultaneous rotation of the sample.
  • the sample is rotated about an axis 3 which is displaced in the direction of the arrow P or in the opposite direction during the course of the measurement.
  • the displacement is parallel to the longitudinal axis z of the lens 5. Accordingly, this axis z and the arrow P representing the displacement direction extend parallel. Accordingly, in the schematic drawing, in the current arrangement a section of the sample denoted 9n would be recorded. This section is located on the focal plane 2 and is perpendicular to the longitudinal axis z of the lens 5. As the data recording continues, further section images 9 (not shown) would be recorded which extend in a spiral as far as the axis of rotation 3.
  • Fig. 3a is a view from below of a sample suspension device 10 having a manipulator 11a, 11b.
  • the actual sample suspension takes place in the centre of the suspension system 12 shown, which is in the form of an insert 12 (not shown) into an annular holder 13, the position of which can be varied very precisely by way of micrometer screws 11a and 11b arranged on the holder 13.
  • the micrometer screws 11a and 11b are arranged at a 90° angle to one another, in such a way that precise positioning is possible in these directions.
  • the two micrometer screws 11a, 11b allow to move the sample 1 relative to the rotational axis of the rotating stage RS of the microscope (not shown in fig. 3a , see fig. 6a ) and to center the sample 1 in the field of view during the entire 360° rotation.
  • Fig. 3b is a side view of the sample suspension device 10 having a manipulator 11a, 11b from Fig. 3a .
  • the ring 13 having the micrometer screws 11a and 11b, which holds the actual sample suspension system 12 and by means of which the position thereof can be set precisely, can be seen in the central region of the drawing.
  • the sample 1 itself is arranged at the lower end of the sample suspension system 12. To rotate the sample, the entire sample suspension system 12 is rotated. For displacing the sample 1 in the longitudinal direction of the lens 5, the sample suspension device 10 including the manipulator ring 13 is displaced.
  • Fig. 3c is a view from diagonally below of the sample suspension device 10 having a manipulator 11 from Fig. 3a .
  • the sample suspension system 12 has an opening 14, in which the sample 1 can be arranged, at least at the lower end. The sample 1 projects out beyond the lower end of the sample suspension system 12.
  • the micrometer screws 11a and 11b arranged mutually perpendicular can be seen to the side of the holding ring 13 for the sample suspension system 12, by means of which the sample suspension system 12 can be precisely positioned.
  • three magnets 18a, 18b, 18c are positioned at the lower surface of the sample holder 13. It is preferred to position at least three magnets in order to mount the sample holder 13 to the rotating stage RS of the imaging chamber IC (see fig. 6a and 6b ).
  • the middle axis of the first one 18a of the magnets 18a, 18b, 18c intersects the extension of the length axis of the manipulator 11b to form a right angle with it.
  • fig. 3a further shows the three magnets 18a, 18b, 18c being arranged on the circumference of a common virtual circle, wherein the diameter of the common virtual circle is in the range of 80-99%, preferably in the range of 85-90% of the outer diameter of the sample holder 13. Furthermore, magnet 18a is spaced apart from the other two magnets 18b, 18c by an angle of 120°. As a result, this applies analogously to each of the other two magnets 18b and 18c.
  • the three magnets 18a, 18b, 18c each show a cylindrically shape with the same diameter.
  • a lower surface of the three magnets 18a, 18b, 18c preferably flushes with the lower surface of the sample holder 13.
  • the sample suspension system 12 itself is shown as a whole in Fig. 3d . It is distinguished by an elongate cylindrical shaft 15 in which the sample 1 is held and can be introduced from above into the coverage region of the lens 5. By way of an extended region, the sample suspension system 12 is mounted in a holding ring 13 (not shown).
  • Fig. 3e is a detail of the sample suspension system 12 having a sample 1 attached thereto.
  • the lower end of the sample suspension system 12 can be seen, from which a transparent tube 16 attached therein protrudes.
  • the sample 1 is located in this tube 16, which may be filled with air, water, a buffer, a nutrient solution or a gel. Because of the transparent tube 16 and the likewise medium located therein, the sample 1 can be optically captured by the lens 5.
  • the material of the transparent tube 16 and the medium located therein preferably have the same or at least a very similar refractive index.
  • Fig. 4a - d show the result of imaging a sample of this type.
  • Fig. 4a is an image of a zebrafish embryo 21 from the incident-light method.
  • Fig. 4b is a side view of a zebrafish embryo 21 from the transmitted-light method, as recorded in accordance with the present invention.
  • the zebrafish embryo 21 was arranged in the sample tube 16 in such a way that the longitudinal axis thereof extended largely parallel to the longitudinal axis of the sample tube 16.
  • the axis of rotation 3 about which the sample 1, 21 was rotated during recording is marked by the line 3 extending in the longitudinal direction of the zebrafish embryo 21.
  • Fig. 4c is a further view of the same zebrafish embryo 21 from the transmitted-light method, but after rotation through 90°.
  • the axis of rotation 3 is also marked by the line 3 extending parallel to the longitudinal orientation of the zebrafish embryo.
  • the lines 22, 23 extending perpendicular to the longitudinal zebrafish embryo in Fig. 4b and 4c represent sections as shown in Fig. 4d and 4e .
  • Fig. 4d is a sectional view of a zebrafish embryo 21 perpendicular to the longitudinal axis thereof.
  • image 4d is not an image recorded by the microscope, but a section calculated from a plurality of images.
  • the microscope exclusively records sections from planes which extend parallel to the axis of rotation 3, such as are shown for example in Fig. 4b and 4c . Because of the very good resolution which can be achieved by this method and the rapid recording process, after calculating the three-dimensional structure it is also possible to have sections 22, 23 perpendicular to the recording axis displayed. As can be seen from image 4d, even a calculated sectional view of this type is low in artefacts and makes possible very precise conclusions as to the positioning of individual objects in the interior of the sample 1, 21.
  • Fig. 4e is likewise a sectional view 22, 23 of a zebrafish embryo 21, but in the region of the head at the level of the eyes.
  • the eyes can be seen as black dots 24a and 24b and can be localised very well.
  • FIG. 5a is a sectional view of a zebrafish embryo 21 by light sheet fluorescence microscopy (LSFM). Fluorescence-marked regions can be captured selectively. However, all other regions of the zebrafish embryo 21 which are not fluorescence-marked are not shown in this method. Thus, in this method, in particular information as to the arrangement of the fluorescence-marked objects in the interior of the embryo is lost. LSFM merely makes it possible to represent all of the fluorescence-marked or fluorescing objects and the relative arrangement thereof.
  • LSFM merely makes it possible to represent all of the fluorescence-marked or fluorescing objects and the relative arrangement thereof.
  • Fig. 5b is a combination of the LSFM-image of Fig. 5a in superpositions of a transmitted-light microscopy sectional view of the same object. As a result of this superposition, it is possible to localise the fluorescence-marked objects in the interior of the zebrafish embryo 21. Conclusions as to the spatial arrangement and the distribution of the fluorescence-marked objects are thus possible.
  • Fig. 5c is a superposed layer view analogous to Fig. 5b but of a different layer.
  • conclusions can be drawn as to the localisation of the fluorescence-marked particles in the interior of the zebrafish embryo 21. For example, no fluorescence-marked particles can be detected in the yolk sac 25, which can be seen in the bottom-left image portion.
  • Fig. 5d is a LSFM view of a layer from the head region of the zebrafish embryo. This image is not an image recorded by the microscope, but rather a sectional view calculated back from the 3D model. As in Fig. 5a , it is not possible to draw conclusions as to the positioning of the individual fluorescence-marked particles in the interior of the zebrafish embryo 21, but rather only the arrangement of the fluorescence-marked objects relative to one another can be determined.
  • Fig. 5e is a superposition of the image of Fig. 5d with a corresponding transmitted-light microscopy layer view.
  • the transmitted-light microscopy layer view is not a layer recorded as an image by the transmitted-light microscope, but rather a layer calculated from the 3D model.
  • a conclusion is possible as to the concentration and localisation thereof relative to the non-fluorescence-marked boundaries of the zebrafish embryo 21.
  • Fig. 5f is a LSFM view of a section through a zebrafish embryo at the level of the yolk sac 25. Since, as stated previously in relation to Fig. 5c , the yolk sac 25 does not contain any fluorescence-marked objects, there is no information on the size and arrangement of the yolk sac 25 in the pure fluorescence view. Any relevant data are suppressed in this recording technique.
  • Fig. 5g is a superposition of the LSFM image of Fig. 5f with a corresponding transmitted-light microscopy sectional view.
  • Fig. 6a shows a schematic of the microscope setup.
  • Each laser beam LB used for bidirectional SPIM illumination is passed through a cylindrical lens CL, a telescope (lenses L1 and L2) and sent to the imaging chamber IC through an illumination lens.
  • Two 45° mirrors M are used to properly align each beam.
  • Brightfield illumination is provided in transmission mode by an LED backlight N.
  • the sample 1 (not shown) dips into the medium-filled imaging chamber IC from the top. It is held and moved by a fast rotational stage RS and a 3-axis linear stage LS.
  • the image of the sample 1 is formed at the detector using a water-dipping objective O (see fig. 6b ) and a tube lens TL.
  • the detected light is passed through a filter wheel FW and a 45° mirror M.
  • Fig. 6b shows a top view of the imaging chamber IC. Illumination and detection are provided by four water-dipping objectives O. Light-sheets illuminate the sample from two opposite sides (beams LB) and brightfield illumination is provided by an LED backlight N, placed at the back of one of the objectives O. The transmitted or fluorescence light is collected by a fourth objective O and passed through the filter wheel FW.

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Claims (12)

  1. Verfahren zur Erstellung eines optischen Tomogramms, dadurch gekennzeichnet, dass es die folgenden Schritte umfasst:
    a) Bereitstellen eines optischen Mikroskops umfassend ein Mikroskopobjektiv,
    b) Anordnen einer Probe (1) in der optischen Abdeckungsregion einer Linse (5) des Mikroskops,
    c) Einstellen des Brennpunkts der Linse auf eine spezielle Brennebene (2),
    d) Aufnehmen eines Bilds der Probe durch das Mikroskop,
    e) Drehen der Probe um einen Winkel α,
    f) Verschieben der Probe entlang der longitudinalen Achse (z) der Linse (5) und/oder senkrecht zu der Ebene des zuvor aufgenommenen Bildes (9) in Abhängigkeit einer Tiefenschärfe des Mikroskopobjektivs,
    g) Fortsetzen des Verfahrens mit Schritt d) bis eine vorbestimmte Zahl von Schnittbildern (9) der Probe (1) aufgenommen worden sind,
    wobei die Probe (1) entlang der longitudinalen Achse (z) der Linse (5) und/oder senkrecht zu der Ebene des zuvor aufgenommenen Bildes (9), in Übereinstimmung mit Schritt f), mindestens einmal während einer Drehung der Probe um 360° verschoben wird, so dass eine signifikante Beschleunigung eines Aufnehmens der Schnittbilder, welche für die optische Tomographie benötigt werden, und eine Reduktion der Belastung der lebenden Exemplare durch Bewegen der Probe entlang einer glatten Trajektorie ohne jegliche abrupte Änderungen in der Richtung realisiert werden, wobei die individuellen Schnittbilder digital aufgenommen und in einem Speicher gespeichert werden und durch ein Datenverarbeitungsgerät kombiniert werden, um ein dreidimensionales Modell zu bilden, welches die innere dreidimensionale Struktur der Probe (1) darstellt, und wobei der Speicher des optischen Mikroskops eine minimale Speicherkapazität aufweist, welche es ermöglicht, mindestens eine Anzahl i von Schnittbildern, welche aufgenommen werden können während einer Drehung der Probe um 360°, zu speichern, wobei i die Anzahl der individuellen Schnittbilder ist, welche während einer kompletten Drehung der Probe aufgenommen werden, und wobei weiter nach Drehung um einen Winkel α ein Schnittbild aufgenommen wird, so dass i 360 α
    Figure imgb0010
     ist, und wobei die Berechnung des dreidimensionalen Modells der Probe (1) erst nach der ersten kompletten Drehung der Probe um 360° startet.
  2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die Probe (5) n Mal von jedem Winkel α aufgenommen worden ist, in verschiedenen Brennebenen (2) in jedem Fall, wobei n größer als 3 ist, bevorzugt größer als 5, bevorzugter zwischen 10 und 1000 und besonders bevorzugt zwischen 15 und 100.
  3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, dass nach jeder Drehung um den Winkel α die Probe (1) um einen Abstand entlang der longitudinalen Achse (z) der Linse (5) und/oder senkrecht zu der Ebene des zuvor aufgenommenen Bildes (9) verschoben wird, wobei der Abstand, welcher einer Länge von 360 ∗ D α ∗ n
    Figure imgb0011
     entspricht, wobei D der gesamten Verschiebung der Probe während der Erstellung des optischen Tomogramms entspricht.
  4. Verfahren nach Anspruch 2, dadurch gekennzeichnet, dass nach einer kompletten Drehung um 360° die Probe (1) um einen Abstand entlang der longitudinalen Achse (z) der Linse (5) und/oder senkrecht zu der Ebene des zuvor aufgenommenen Bilds (9) verschoben wird, wobei der Abstand einer Länge von D n
    Figure imgb0012
     entspricht, wobei D der gesamten Verschiebung der Probe während der Erstellung eines optischen Tomogramms entspricht.
  5. Verfahren nach entweder Anspruch 1 oder Anspruch 2, dadurch gekennzeichnet, dass die Probe (1) stetig entlang der longitudinalen Achse (z) der Linse (5) und/oder senkrecht zu der Ebene des zuvor aufgenommenen Bildes (9) verschoben wird während der Aufnahme von mindestens drei Bildern in verschiedenen Winkeln a.
  6. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Linse (5) im Wesentlichen horizontal angeordnet ist.
  7. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Probe (1) in den Abdeckungsbereich der Linse (5) herunterhängt, um die Probe (1) in dem optischen Abdeckungsbereich der Linse anzuordnen.
  8. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass vor der ersten Aufnahme die Probe (1) genau positioniert ist, um auf der Rotationsachse in dem optischen Abdeckungsbereich der Linse (5) des Mikroskops zu liegen, bevorzugt mittels Mikrometerschrauben (11), welche auf der Aufhängevorrichtung (10) der Probe angeordnet sind.
  9. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass ein Hellfeldmikroskop als das optische Mikroskop verwendet wird.
  10. Optisches Mikroskop, welches dazu ausgebildet und vorgesehen ist, den Verfahrensanspruch 1 zum Erzeugen von Tomogrammen auszuführen, was mindestens eine Linse (5), mindestens eine Aufhängevorrichtung (10) für die Probe und mindestens ein Beleuchtungsgerät (6) umfasst, dadurch gekennzeichnet, dass die Aufhängevorrichtung (10) der Probe
    a) drehbar um eine Achse (3) ist, welche senkrecht zu der longitudinalen Achse (z) der Linse ist und
    b) verschiebbar entlang der longitudinalen Achse (z) der Linse (5) ist,
    wobei die individuellen Bilder digital aufgenommen und in einem Speicher gespeichert werden und durch ein Datenverarbeitungsgerät kombiniert werden, um ein dreidimensionales Modell zu bilden, welches die innere dreidimensionale Struktur der Probe (1) darstellt, und wobei der Speicher des optischen Mikroskops eine minimale Speicherkapazität aufweist, welche es ermöglicht, mindestens eine Anzahl i von Schnittbildern, welche aufgenommen werden können während einer Drehung der Probe um 360°, zu speichern, wobei i die Anzahl der individuellen Schnittbilder ist, welche während einer kompletten Drehung der Probe aufgenommen werden, und wobei weiter nach Drehung um einen Winkel α ein Schnittbild aufgenommen wird, so dass i 360 α
    Figure imgb0013
     ist, und wobei die Berechnung des dreidimensionalen Modells der Probe (1) erst nach der ersten kompletten Drehung der Probe um 360° startet.
  11. Optisches Mikroskop nach Anspruch 12, dadurch gekennzeichnet, dass es eine Datenverarbeitungsvorrichtung umfasst, welche den Speicher aufweist oder mit einem verbunden ist, mittels welcher Vorrichtung digital aufgenommene individuelle Bilder durch das Mikroskop speicherbar sind und anschließend kombiniert werden, um ein dreidimensionales Modell der Probe (1) zu erzeugen.
  12. Optisches Mikroskop nach einem der Ansprüche 10 bis 11, dadurch gekennzeichnet, dass es ein Hellfeldmikroskop ist und mindestens ein Beleuchtungsgerät (6) auf der Seite der Probe (1) gegenüber der Linse (5) angeordnet ist.
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